Pharmaceutical compositions comprising small interfering RNA inhibitors of NOX3

ABSTRACT

This invention relates to a method of identifying a modulator of an NADPH oxidase, whereby said modulator is suitable as a lead compound and/or as a medicament for the treatment and/or prevention of hearing loss and/or phantom hearing, the method comprising the steps of (a) contacting a test compound with a protein, wherein said protein (i) comprises or consists of the amino acid sequence of any one of SEQ ID NO: 1, 3 or 5, or (ii) is encoded by a nucleic acid comprising or consisting of the sequence of any one of SEQ ID NO: 2, 4, 6, 23 or 24, or (iii) is a fragment of the protein according to (i) or (ii) and exhibits NADPH oxidase activity, or (iv) has a sequence at least 75% identical with the protein according to (i) or (ii) or with the fragment according to (iii) and exhibits NADPH oxidase activity, and optionally with one or more NADPH oxidase subunits, under conditions allowing binding of said test compound to said protein or, if present, said subunit(s); (b) optionally determining whether said test compound binds to said protein or, if present, said subunit(s); and (c) determining whether (ca) said test compound, upon contacting in step (a); or (cb) said test compound, upon binding in step (b) modulates the expression and/or activity of said protein or, if present, said subunit(s). Also provided are pharmaceutical compositions, medical uses and diagnostic uses of compounds of the invention.

This application is a divisional of co-pending U.S. application Ser. No.13/336,147, filed Dec. 23, 2011, which is a divisional of U.S.application Ser. No. 11/628,419 filed Oct. 3, 2007, now U.S. Pat. No.8,088,356, which is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/EP2005/006061 filed Jun. 6, 2005,which claims priority to European Application No. 04013266.4 filed Jun.4, 2004, the entire text and figures of which disclosures areincorporated herein by reference without disclaimer.

The sequence listing that is contained in the file named“UGENP0012USD2_ST25.txt”, which is 219 KB (as measured in MicrosoftWindows®) and was created on Apr. 2, 2014, is filed herewith byelectronic submission and is incorporated by reference herein.

This invention relates to a method of identifying a modulator of anNADPH oxidase, whereby said modulator is suitable as a lead compoundand/or as a medicament for the treatment and/or prevention of hearingloss and/or phantom hearing, the method comprising the steps of (a)contacting a test compound with a protein, wherein said protein (i)comprises or consists of the amino acid sequence of any one of SEQ IDNO: 1, 3 or 5, or (ii) is encoded by a nucleic acid comprising orconsisting of the sequence of any one of SEQ ID NO: 2, 4, 6, 23 or 24,or (iii) is a fragment of the protein according to (i) or (ii) andexhibits NADPH oxidase activity, or (iv) has a sequence at least 75%identical with the protein according to (i) or (ii) or with the fragmentaccording to (iii) and exhibits NADPH oxidase activity, and optionallywith one or more NADPH oxidase subunits, under conditions allowingbinding of said test compound to said protein or, if present, saidsubunit(s); (b) optionally determining whether said test compound bindsto said protein or, if present, said subunit(s); and (c) determiningwhether (ca) said test compound, upon contacting in step (a); or (cb)said test compound, upon binding in step (b) modulates the expressionand/or activity of said protein or, if present, said subunit(s). Alsoprovided are pharmaceutical compositions, medical uses and diagnosticuses of compounds of the invention.

In this specification, a number of documents is cited. The disclosure ofthese documents, including manufacturer's manuals, is herewithincorporated by reference in its entirety.

Hearing impairment is a widespread and severe sensory deficit. It is thethird most prevalent major chronic disability in the over 65-year-oldage group, but also found in younger persons. Slightly more than 1percent of people under the age of 17 have hearing loss, the prevalencerises to 12 percent between the ages of 45 and 64, to 24 percent betweenthe ages of 65 and 74, and up to 39 percent for ages over 75. There arethree major causes of hearing loss: noise-dependent hearing loss,drug-associated hearing loss and age-associated hearing loss.Interestingly, there appears to be a common mechanism to three majorcauses of hearing loss, namely destruction of sensory epithelium andcochlear neurons through reactive oxygen species. In terms of treatment,no efficient drug treatment or prophylaxis of hearing loss are availableat this point and the only option at present is the use of hearing aids.This situation is further aggravated by the limited understanding of themolecular processes involved in hearing loss and the scarcity ofsuitable molecular targets for therapeutic intervention.

The inner ear is a highly complex structure involved in hearing andbalancing. The conversion of sound into electrical signals occurs withinthe cochlea, in the organ of Corti, and the electrical signals areconducted by the axons of spiral ganglion neurons to the brain. Thelinear movement of the head is sensed by the otolith organs (utricle andsaccule) and the rotation movements by the ampullas of the semicircularcanals. The signals generated in the vestibular system are transmittedby the vestibular ganglion neurons to the central nervous system.

Hearing impairment due to loss of cochlear function occurs frequently,if not invariably over lifetime. Noise and ototoxic chemicals may leadto a precocious, rapid hearing loss, while age itself leads to a moreinsidious, chronic loss of hearing. Research over the last decades hasidentified reactive oxygen species (ROS¹) as the major factor mediatinghearing loss [1]. ROS is generated within the cochlea after exposure toototoxic drugs (e.g. cisplatin [2, 3], aminoglycoside antibiotics [3])or to noise [4]. Signs of oxidative stress, such as DNA damage and lipidperoxidation, have been documented in vivo in response to thosechallenges [5, 6], as well as in cochlear aging [7]. The vestibularsystem is also damaged by ototoxic drugs [8, 9] in a process thatincludes excessive ROS production [10, 11]. ¹ The abbreviations usedare: bp, base pair; DPI, diphenylene iodonium; DUOX, dual domainoxidase; 5-FU, 5-Fluorouracil; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; gp91^(phox), 91-kDa glycoprotein subunit of the phagocyteNADPH oxidase; NOX, NADPH oxidase; NOXA1, NOX activator 1; NOXO1, NOXorganizer 1; PMA, phorbol 12-myristate 13-acetate; PCR, polymerase chainreaction; ROS, reactive oxygen species; RT-PCR, reversetranscription-PCR; SOD, superoxide dismutase.

While the role of oxidative stress in inner ear damage is wellestablished, its source is poorly understood. A role of non-enzymaticgeneration of ROS by ototoxic compounds has been suggested [12]. Thepossibility that a superoxide-generating enzyme could be localizedwithin the inner ear, and thereby account for the oxidative damage ofthis organ, has received little attention.

Over the last decade, it has been proven that the expression ofsuperoxide-generating NADPH oxidases is not restricted to phagocytes.Beside the well-known catalytic subunit of the phagocyte NADPH oxidase,gp91^(phox)/NOX2 (for review see [13]), six other superoxide-producingenzymes have been identified in mammals [14, 15]. For most NOX and DUOXenzymes, a predominant tissue localization has been described, e.g.colon epithelium for NOX1 [16, 17], kidney cortex for NOX4 [18],lymphoid organs and testis for NOX5 [19], and the thyroid gland forDUOX1 and DUOX2 [20, 21]. For NOX3, with the exception of some very lowlevel expression in the embryonic kidney [22], no convincing tissuelocalization had been found so far.

Our knowledge of the activation mechanisms of members of the NOX/DUOXfamily varies considerably among individual enzymes. NOX1 andgp91^(phox)/NOX2 are subunit-dependent enzymes that need to assemblewith an activator subunit (NOXA1 and p67^(phox), respectively) and anorganizer subunit (NOXO1 and p47^(phox), respectively) to generatesuperoxide [23-26]. NOX5, DUOX1 and DUOX2, on the other hand, haveN-terminal Ca²⁺-binding motifs (EF-hand domains), and so far one ofthem, NOX5, has been shown to be activated by increased Ca²⁺concentration [27]. The mechanism of NOX4 activation is less clear.There are indications that it might be a constitutively active enzyme[18].

Tinnitus, also referred to as phantom hearing, is a common and in someinstances invalidating medical complaint. Presently, the pathophysiologyof the disease is poorly understood and there is not proven causativetreatment available. There is however evidence that reactive oxygenspecies might play a role in the pathophysiology of tinnitus (Neri S.Tinnitus and oxidative stress in a selected series of elderly patients.Arch Gerontol Geriatr. 2002; 35 Suppl:219-23) and there are at leastsome reports that suggest a beneficial effect of antioxidant medicationsuch as Gingko extract on the course of the disease (e.g. Schneider D etal. Gingko biloba (Rokan) therapy in tinnitus patients and measurableinteractions between tinnitus and vestibular disturbances. Int TinnitusJ. 2000; 6(1):56-62). Thus, NOX3 might also be involved in thepathophysiology of tinnitus and the use of a NOX3 modulator or inhibitoris an interesting new concept for the treatment of tinnitus.

US-A1 20040001818 and WO-A1 0230453 describe methods of inhibitingangiogenesis, endothelial cell migration or endothelial cellproliferation using NADPH oxidase inhibitors.

EP-A2 1410798 describes a pharmaceutical composition comprising and usesof inhibitors of the production or the release of reactive oxygenmetabolites (ROMs) and of compounds effective to scavenge ROMs. The usesare directed to the manufacture of a medicament for the treatment ofAdult Respiratory Distress Syndrome (ARDS); ischemia or reperfusioninjury, infectious disease, autoimmune or inflammatory diseases, andneurodegenerative diseases. Compounds effective to inhibit enzymatic ROMproduction or release comprise NADPH oxidase inhibitors.

EP-A2 0914821 relates to a method for diagnosis of atherosclerosisinvolving measurement of NADPH oxidase activity.

WO-A2 9719679 describes the use of NADPH oxidase inhibitors for themanufacture of a medicament for prevention of atherosclerosis.

US-A1 20040009901 relates to a method of treating a mammal having anautoimmune condition involving NADPH oxidase deficiency. Also, a methodfor identifying an agent that enhances NADPH oxidase activity isdescribed.

WO-A2 02079224 relates to human peptides and proteins that are relatedto NADPH oxidase subfamily and methods for identifying modulatorsthereof. The proteins are described as being substantially similar top47phox.

WO-A2 04007689 describes regulatory proteins for Nox enzymes, which arereferred to as p41Nox proteins, and nucleic acid sequences encodingthese proteins. Furthermore, a method for identifying a compound thatmodulates superoxide production is rescribed, the method involvingadministration of the protein. The envisaged medical indications relateto abnormal cell growth and proliferation and include cancer, prostatichypertrophy and atherosclerosis.

NCBI Entrez protein database entry NP_056533 comprises the amino acidsequence of human NADPH oxidase 3 (NOX3). The sequence is 568 aminoacids in length. The database entry recites similarity to gp91phox.

In view of the limited understanding of processes leading to hearingloss and phantom hearing, the technical problem underlying the presentinvention was therefore the provision of means and methods for thedevelopment of drugs for treatment of hearing loss and phantom hearing.

Accordingly, this invention relates to a method of identifying amodulator of an NADPH oxidase, whereby said modulator is suitable as alead compound and/or as a medicament for the treatment and/or preventionof hearing loss and/or phantom hearing, the method comprising the stepsof (a) contacting a test compound with a protein, wherein said protein(i) comprises or consists of the amino acid sequence of any one of SEQID NO: 1, 3 or 5, or (ii) is encoded by a nucleic acid comprising orconsisting of the sequence of any one of SEQ ID NO: 2, 4, 6, 23 or 24,or (iii) is a fragment of the protein according to (i) or (ii) andexhibits NADPH oxidase activity, or (iv) has a sequence at least 75%identical with the protein according to (i) or (ii) or with the fragmentaccording to (iii) and exhibits NADPH oxidase activity, and optionallywith one or more NADPH oxidase subunits, under conditions allowingbinding of said test compound to said protein or, if present, saidsubunit(s); (b) optionally determining whether said test compound bindsto said protein or, if present, said subunit(s); and (c) determiningwhether (ca) said test compound, upon contacting in step (a); or (cb)said test compound, upon binding in step (b) modulates the expressionand/or activity of said protein or, if present, said subunit(s).

The term “modulator” designates a compound modulating the activity of atarget molecule, preferably by performing one or more of the followingeffects: (i) the transcription of the gene encoding the protein to bemodulated is modulated, (ii) the translation of the mRNA encoding theprotein to be modulated is modulated, (iii) the protein performs itsbiochemical function with modulated efficiency in presence of themodulator, and (iv) the protein performs its cellular function withmodulated efficiency in presence of the modulator. It is understood thatthe term “modulator” includes inhibitors and activators at allregulatory levels mentioned above.

The term “NADPH oxidase” comprises any NADPH oxidase. It includes NOXenzymes such as NOX1, NOX2, NOX3, NOX4 and NOX5 as well as DUOX enzymessuch as DUOX1 and DUOX2 (see references 13 to 27).

The term “lead compound” designates a compound which is a drug candidateand which may require chemical modifications in order to optimize itspharmacological properties and eventually become a drug to be formulatedas a medicament. Methods of optimization are known in the art andfurther detailed below.

The term “hearing loss” according to the invention embraces drug-,noise- and age-related hearing loss. Age-related hearing loss is alsoreferred to as presbyacusis. The term “phantom hearing”, also known as“tinnitus”, is a common and in some instances invalidating medicalcomplaint.

The term “protein” recited in the main claim extends to homologueshaving at least 75% sequence identity. Preferably, the sequence identitylevel is 80% or 85%, more preferred 90% or 95%, and yet more preferred98% or 99%. For the purpose of determining the level of sequenceidentity, two nucleotide or protein sequences can be alignedelectronically using suitable computer programs known in the art. Suchprograms comprise BLAST (Altschul et al. (1990), J. Mol. Biol. 215,403-410), variants thereof such as WU-BLAST (Altschul & Gish (1996),Methods Enzymol. 266, 460-480), FASTA (Pearson & Lipman (1988), Proc.Natl. Acad. Sci. USA 85, 2444-2448) or implementations of theSmith-Waterman algorithm (SSEARCH, Smith & Waterman (1981), J. Mol.Biol. 147, 195-197). These programs, in addition to providing a pairwisesequence alignment, also report the sequence identity level (usually inpercent identity) and the probability for the occurrence of thealignment by chance (P-value). Programs such as CLUSTALW (Higgins et al.(1994), Nucleic Acids Res. 22, 4673-4680) can be used to align more thantwo sequences.

The optional presence of one or more NADPH oxidase subunits relatesinter alia to embodiments, wherein not only modulators exerting theireffect exclusively directly on the NADPH oxidase are to be identified,but also modulators which act by interfering with the association of theNADPH oxidase with said subunit(s) are to be identified. Such modulatorsmay be compounds binding to regions of the NADPH oxidase and/or of thesubunit(s) involved in subunit association. In other words, a testcompound identified by the method of the invention which interferes withassociation (e.g. binds to regions of the NADPH oxidase and/or of thesubunit(s) involved in subunit association) is an example of a testcompound according to the invention which either modulates expressionand/or activity of the protein defined in the main embodiment ormodulates the expression and/or activity of said subunit(s).

Also embraced by the invention is a method as defined above, whereintest compounds may be identified which modulate the expression and/oractivity of both the protein defined in the main embodiment and saidsubunits.

In the following, the interactions of an NADPH oxidase with its subunitsis exemplified for the NADPH oxidase 3 (NOX3). NOX3 activity requiresthe widely distributed membrane NOX subunit p22^(phox). However, in theabsence of further, viz. cytoplasmic subunits, no high level, but onlylow level ROS generation occurs. In contrast in the presence of thecombination of one activator subunit (either NOXA1 or p67phox/NOXA2) andone organizer subunit (either NOXO1 or p47^(phox)/NOXO2) NOX3 is capableof generating high levels of ROS. In addition, the NOX3 activity mostlikely also involves the ubiquitous GTP-binding protein Rac. Theinteraction sites between the partners are depicted in the followingscheme.

A key interaction is the binding of the activator domain of theactivator subunits (amino acids 202-212 for hNOXA1 and amino acids200-210 for hp67^(phox)/NOXA2) to NOX3. It is not clear whether there isa direct interaction of the organizer subunits with NOX3, but there isan indirect interaction with NOX3 through binding to p22^(phox) via thetandem SH3 domain (amino acids 158-217 and 233-289 for hNOXO1 and aminoacids 156-216 and 226-286 for hp47^(phox)/NOXO2) and through binding toan SH3 domain of the activator subunit (amino acids 402-463 for hNOXA1and amino acids 457-513 for hp67^(phox)/NOXA2) through its proline-richregion (amino acids 321-331 for hNOXO1 and 360-370 forhp47^(phox)/NOXO2). The precise site of interaction between NOX3 andp22^(phox), as well as the sites of interaction of Rac1 with NOX3 andthe activator subunits (NOXA1 or p67^(phox)) are not known. The tablebelow provides a compilation of the interaction sites.

binding region of the subunit target activator region of activatorsubunit NOX3 (aa 202-212 for hNOXA1 and aa 200-210 forhp67^(phox)/NOXA2) tandem SH3 domain of organizer p22^(phox) subunit (aa158-217 and aa 233-289 for hNOXO1 and aa 156-216 and 226-286 forhp47^(phox)/NOXO2) proline-rich region of organizer subunit SH3 domainof activator subunit (aa 321-331 for hNOXO1 and (aa 402-463 for hNOXA1and 360-370 for hp47^(phox)/NOXO2) aa 457-513 for hp67^(phox)/ NOXA2)

The optional determination of binding test compounds in step (b) relatesto any biophysical binding assay, which may be used to identify bindingtest molecules prior to performing the functional assay with the bindingtest molecules only. Suitable biophysical binding assays are known inthe art and comprise fluorescence polarization (FP) assay, fluorescenceresonance energy transfer (FRET) assay and surface plasmon resonance(SPR) assay. Step (b) is particularly advantageous if said biophysicalassay is more amenable to high throughput than the functional assay.

Step (c) relates to the above mentioned functional assay. Determiningwhether a test compound, or a binding test compound, modulates theexpression of a target protein may be accomplished by measuring theexpression level. In a more preferred embodiment, the expression levelto be determined is the mRNA expression level. Methods for thedetermination of mRNA expression levels are known in the art andcomprise Real Time PCR, Northern blotting and hybridization onmicroarrays or DNA chips equipped with one or more probes or probe setsspecific for transcripts encoding proteins of the NADPH oxidase family.

In another more preferred embodiment, the expression level to bedetermined is the protein expression level. The skilled person is awareof methods for the quantitation of proteins. Amounts of purified proteinin solution can be determined by physical methods, e.g. photometry.Methods of quantifying a particular protein in a mixture rely onspecific binding, e.g. of antibodies. Specific detection andquantitation methods exploiting the specificity of antibodies compriseimmunohistochemistry (in situ) and surface plasmon resonance. Westernblotting combines separation of a mixture of proteins by electrophoresisand specific detection with antibodies.

The present invention also relates to a method of identifying amodulator of an NADPH oxidase, whereby said modulator is suitable as alead compound and/or as a medicament for the treatment and/or preventionof hearing loss and/or phantom hearing, the method comprising the stepsof (a) contacting a test compound with a protein, wherein said protein(i) comprises or consists of the amino acid sequence of any one of SEQID NO: 1, 3 or 5, or (ii) is encoded by a nucleic acid comprising orconsisting of the sequence of any one of SEQ ID NO: 2, 4, 6, 23 or 24,or (iii) is a fragment of the protein according to (i) or (ii) andexhibits NADPH oxidase activity, or (iv) has a sequence at least 75%identical with the protein according to (i) or (ii) or with the fragmentaccording to (iii) and exhibits NADPH oxidase activity, under conditionsallowing binding of said test compound to said protein; and (b)determining whether said test compound, upon contacting in step (a)modulates the expression and/or activity of said protein.

The present invention also relates to a method of identifying amodulator of an NADPH oxidase, whereby said modulator is suitable as alead compound and/or as a medicament for the treatment and/or preventionof hearing loss and/or phantom hearing, the method comprising the stepsof (a) contacting a test compound with a protein, wherein said protein(i) comprises or consists of the amino acid sequence of any one of SEQID NO: 1, 3 or 5, or (ii) is encoded by a nucleic acid comprising orconsisting of the sequence of any one of SEQ ID NO: 2, 4, 6, 23 or 24,or (iii) is a fragment of the protein according to (i) or (ii) andexhibits NADPH oxidase activity, or (iv) has a sequence at least 75%identical with the protein according to (i) or (ii) or with the fragmentaccording to (iii) and exhibits NADPH oxidase activity, under conditionsallowing binding of said test compound to said protein; (b) determiningwhether said test compound, upon contacting in step (a) modulates theexpression and/or activity of said protein; and (c) performing clinicaltrials with said modulator.

In a preferred embodiment of the method of the invention, saidcontacting comprises contacting with one or more NADPH oxidase subunits,under conditions allowing binding of said test compounds to saidsubunit(s), and wherein said determining comprises determining whethersaid test compound modulates the expression and/or activity of saidsubunit(s).

In a further preferred embodiment the method further comprises, prior tostep (b), the step of (b′) determining whether said test compound bindsto said protein or, if present, said subunit(s), wherein saiddetermining in step (b) is effected upon binding in step (b′). Themethod according to this preferred embodiment comprises both determiningof whether a test compound, upon contacting in step (a), modulatesexpression and/or activity and the determining of whether a testcompound, upon binding in step (b′), modulates expression and/oractivity. The term “expression and/or activity” relate to, as definedherein above, the expression and/or activity of the protein as definedin the main embodiment and/or of said subunit(s).

Quantitation of the modulation of the activity of an NADPH oxidase maybe effected by quantifying the reactive oxygen species production.Accordingly, said modulation preferably involves modulating the ROSproduction of said protein, and determining in step (c) comprisesquantifying ROS production. Methods of quantifying ROS are known in theart and are further exemplified in Example 4 enclosed herewith.

The inventors for the first time demonstrated high-level expression ofthe NADPH oxidase NOX3 in the inner ear. Thereby, a protein suitable asa target for therapeutic intervention in hearing loss and phantomhearing is provided.

Vestibular and cochlear sensory epithelia develop from a commonectodermal thickening at the head region, called placode [34]. The oticplacode also gives rise to the neurons that will form the inner earganglia [35]. The data presented in the Examples and Figures enclosedherewith suggest that the expression of NOX3 mRNA may follow thispattern.

Furthermore, the inventors demonstrated for the first time that NOX3 isa superoxide-generating enzyme. It is also demonstrated that the patternof subunit- and stimulus-dependence that is distinct from other knownNOX family NADPH oxidases. NOX3, as opposed to NOX1 and NOX2, produceslow levels of superoxide upon PKC activation without the need ofsubunits. While the activation of phagocyte NADPH oxidase is thought tooccur through PKC-dependent phosphorylation of p47^(phox) [13], this,obviously, cannot be the mechanism of the subunit-independent activationof NOX3. At this point, there are numerous possible pathways how PKCmight activate NOX3 (e.g. direct phosphorylation of NOX3, activation ofthe small GTPase protein Rac1, or changes in the lipid environment). Thesubunit-independent ROS-generation by NOX3 is of low level in thetransfected cells. Given the localization of NOX3 in the inner ear,close to or within highly ROS-sensitive cells, it is tempting tospeculate that low, rather than high level superoxide generation is thedefault mode of NOX3 function.

However, NOX3 activity can be massively enhanced by known NOX organizerand regulator/activator subunits. Searches of mouse and human genomicdatabases suggest that there are probably no other close homologues ofp47^(phox) and p67^(phox) than NOXO1 and NOXA1, respectively. Thus, ifNOX3 functions in a subunit-dependent manner in vivo, it would have touse subunits of other NOX enzymes. Based on PCR data shown in FIG. 2,NOX3 could potentially interact with NOXA1 and/or p47^(phox) in theinner ear. However, it cannot be excluded that, under specificcircumstances or in a very limited number of cells, other NOX subunitsmay also be expressed in the inner ear.

Therefore, in a preferred embodiment, said NADPH oxidase subunit(s)is/are the activating subunit(s) NOXA1 and/or p67^(phox)/NOXA2, and/orthe organising subunit(s) NOXO1 and/or p47^(phox)/NOXO2.

In a further preferred embodiment said protein and, if present, saidsubunit(s) is/are comprised in a membrane preparation. Membranepreparations according to the invention may be membrane fractionsobtained, for example, by centrifugation upon cell disruption.Alternatively, said membrane preparation is obtained by reconstitutingthe protein(s) according to the main embodiment with membrane- ormicelle-forming amphiphilic lipids.

In a further preferred embodiment said protein and, if present, saidsubunit(s) is/are comprised in a cell transfected with a nucleic acidencoding said protein. This embodiment relates to a cellular screen.

In a further preferred embodiment of the method of the invention, saidprotein and, if present, said subunit(s) is/are comprised in a non-humananimal. This embodiment relates to an in vivo screen. While lessamenable to high throughput, the in vivo screen offers the advantage ofthe assessment of the disease state of the non-human animal.Accordingly, in a more preferred embodiment, the modulation of ROSproduction involves improving the hearing of said animal and determiningin step (c) involves quantifying said hearing.

In a further preferred embodiment, prior to said contacting, (a′) anototoxic agent and/or an agent increasing the activity and/or theexpression of said protein or subunit(s), is brought into contact withsaid protein or subunit(s) is/are administered to said cell or saidanimal. Administration of an ototoxic agent and/or an agent increasingthe activity and/or the expression of said protein or subunit(s) may beused as a means of modelling (at the cellular level), orinducing/enhancing (at the organismic level) the disease ordisease-related conditions.

Interestingly, while there is almost no literature on the physiologicalfunction of ROS in the inner ear, there is a considerable number ofstudies on the pathological effect of excessive ROS production in thisorgan (for reviews see [1] and [4]). It has been shown in severalpublications that specific ototoxic drugs (such as platinum derivativesor aminoglycoside antibiotics) lead to accumulation of ROS in both thecochlea [3] and the vestibular system [8, 11, 36], and noise trauma hasbeen demonstrated to be a prominent cause of ROS production in thecochlea [37]. A permanent increase of ROS concentration, in turn, leadsprimarily to the death of sensory epithelial cells, and, to a lesserextent, to the death of innervating neurons [1]. Based on the surprisingobservations presented herein and relating to its localization and itscapacity to generate ROS, NOX3 is likely to be a major source of ROS inthe inner ear. The unexpected observation that cisplatin markedlyenhances NOX3-dependent superoxide production, evokes the possibilitythat NOX3 is a mediator of cisplatin-dependent ototoxicity. Time courseand dose-response of the cisplatin-dependent NOX3 activation iscompatible with the time course [2] and dose-response [38] of cisplatintoxicity to inner ear sensory cells.

In a more preferred embodiment, said ototoxic agent is selected from thegroup consisting of salicylates, non-steroidal antiinflammatories,antibiotics, diuretics, cytostatics, quinine derivatives andgastroprotective drugs.

Salicylates include Aspirine and methyl-salicylates.

Non-steroidal antiinflammatories include diclofenac, etocolac,fenprofen, ibuprofen, indomethacin, naproxen, piroxicam and sulindac.

Preferred antibiotics are aminoglycosides such as amikacin, gentamycin,kanamycin, neomycin, netilmicin, streptomycin and tobramycin. Furtherpreferred antibiotics include erythromycin, vancomycin, minocycline,polymixin B, amphotericin B and capreomycin.

Exemplary diuretics according to the invention are bendroflumethazide,bumetadine, chlorthalidone, ethacrynic acid and furosemide.

Cytostatics, or antineoplastic drugs according to the invention includebleomycine, bromocriptine, carboplatinum, cisplatin, methotrexate,nitrogen mustard, vinblastin and vincristine.

Quinine derivatives, being used as antimalarial and antiarrhythmicdrugs, include chloroquine phosphate, quinacrine hydrochloride andquinine sulphate.

Misoprostol is among the envisaged gastroprotective drugs.

In a preferred embodiment of the method of the invention, said NADPHoxidase is NOX3. In a further preferred embodiment said NADPH oxidase isthe protein defined in claim 1.

In a further preferred embodiment, the method of the invention furthercomprises the step of formulating said modulator with a pharmaceuticallyacceptable carrier. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type.

More preferred, and prior to said formulating, the affinity, specificityand/or pharmacological properties of the modulator are optimized and/orclinical trials are performed with said modulator or the optimizedmodulator.

Accordingly, the present invention also relates to a method ofidentifying a modulator of an NADPH oxidase, whereby said modulator issuitable as a lead compound and/or as a medicament for the treatmentand/or prevention of hearing loss and/or phantom hearing, the methodcomprising the steps of (a) contacting a test compound with a protein,wherein said protein (i) comprises or consists of the amino acidsequence of any one of SEQ ID NO: 1, 3 or 5, or (ii) is encoded by anucleic acid comprising or consisting of the sequence of any one of SEQID NO: 2, 4, 6, 23 or 24, or (iii) is a fragment of the proteinaccording to (i) or (ii) and exhibits NADPH oxidase activity, or (iv)has a sequence at least 75% identical with the protein according to (i)or (ii) or with the fragment according to (iii) and exhibits NADPHoxidase activity, under conditions allowing binding of said testcompound to said protein; (b) determining whether said test compound,upon contacting in step (a) modulates the expression and/or activity ofsaid protein; and (c) performing clinical trials with said modulator.

Methods for the optimization of the pharmacological properties ofcompounds identified in screens, generally referred to as leadcompounds, are known in the art and comprise a method of modifying acompound identified as a lead compound to achieve: (i) modified site ofaction, spectrum of activity, organ specificity, and/or (ii) improvedpotency, and/or (iii) decreased toxicity (improved therapeutic index),and/or (iv) decreased side effects, and/or (v) modified onset oftherapeutic action, duration of effect, and/or (vi) modifiedpharmacokinetic parameters (resorption, distribution, metabolism andexcretion), and/or (vii) modified physico-chemical parameters(solubility, hygroscopicity, color, taste, odor, stability, state),and/or (viii) improved general specificity, organ/tissue specificity,and/or (ix) optimized application form and route by (i) esterificationof carboxyl groups, or (ii) esterification of hydroxyl groups withcarbon acids, or (iii) esterification of hydroxyl groups to, e.g.phosphates, pyrophosphates or sulfates or hemi succinates, or (iv)formation of pharmaceutically acceptable salts, or (v) formation ofpharmaceutically acceptable complexes, or (vi) synthesis ofpharmacologically active polymers, or (vii) introduction of hydrophilicmoieties, or (viii) introduction/exchange of substituents on aromates orside chains, change of substituent pattern, or (ix) modification byintroduction of isosteric or bioisosteric moieties, or (x) synthesis ofhomologous compounds, or (xi) introduction of branched side chains, or(xii) conversion of alkyl substituents to cyclic analogues, or (xiii)derivatisation of hydroxyl group to ketales, acetales, or (xiv)N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannichbases, imines, or (xvi) transformation of ketones or aldehydes toSchiff's bases, oximes, acetales, ketales, enolesters, oxazolidines,thiozolidines or combinations thereof; said method optionally furthercomprising the steps of the above described methods.

The various steps recited above are generally known in the art. Theyinclude or rely on quantitative structure-action relationship (QSAR)analyses (Kubinyi, “Hausch-Analysis and Related Approaches”, VCH Verlag,Weinheim, 1992), combinatorial biochemistry, classical chemistry andothers (see, for example, Holzgrabe and Bechtold, Deutsche ApothekerZeitung 140(8), 813-823, 2000).

Individuals to be selected for said clinical trials comprise healthyindividuals, individuals with a disposition or at risk to develophearing loss or phantom hearing and patients suffering from hearing lossor phantom hearing. Hearing loss is understood to comprise drug-, noise-and age-related hearing loss.

Moreover, the present invention also relates to a pharmaceuticalcomposition comprising (a) an antibody, aptamer, or a fragment orderivative thereof binding specifically the protein defined in the mainembodiment; (b) an antisense nucleic acid, an siRNA, or a ribozymebinding specifically a nucleic acid encoding said protein; (c) aiodonium derivative and/or a substituted catechol such as apocynin; (d)a compound comprising the fragment of SEQ ID NO: 11 from position 202 toposition 212, the fragment of SEQ ID NO: 11 from position 402 toposition 463, the fragment of SEQ ID NO: 15 from position 200 toposition 210, the fragment of SEQ ID NO: 15 from position 457 toposition 513, the fragment of SEQ ID NO: 7 from position 158 to position217, the fragment of SEQ ID NO: 7 from position 233 to position 289, thefragment of SEQ ID NO: 7 from position 321 to position 331, the fragmentof SEQ ID NO: 19 from position 156 to position 216, the fragment of SEQID NO: 19 from position 226 to position 286, the fragment of SEQ ID NO:19 from position 360 to position 370; and/or (e) a nucleic acidcomprising a sequence encoding any of the fragments according to (d).The fragments according to (d) are regions of the sequences of therespective SEQ ID NOs known or expected to be involved in subunitassociation.

Said compounds according to (d) may furthermore comprise acell-penetrating peptide. The term “cell-penetrating peptide” relates toa peptide which is capable of entering into cells. This capability maybe exploited for the delivery of fragments defined in (d) to cells.

For example, said compounds may be peptides or polypeptides comprisingboth a fragment as defined in (d) above and a cell-penetrating peptide.Alternatively, other means of functionally linking a fragments asdefined in (d) and a cell-penetrating peptide are envisaged. Preferably,said compounds comprising both a fragment as defined in (d) above and acell-penetrating peptide act as dominant negative cell-permeatinginhibitors.

Said cell-penetrating peptides according to the invention includeTat-derived cell-penetrating peptides [46, 47], Antennapedia peptides orpenetratins [48, 49] such as the peptide having the sequenceArg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys (SEQ IDNO: 25), peptides derived from HSV-1 VP22 [50], transportans [51], MAPpeptides [52] such as the peptide with the sequence KLALKLALKALKAALKLA(SEQ ID NO: 26), signal sequence-based cell-penetrating peptides (NLS)[53], hydrophobic membrane translocating sequence (MTS) peptides [53]and arginine-rich transporters for drugs. According to an overview ofcell-penetrating peptides is provided in [45], CPPs are divided into twoclasses: the first class consists of amphipathic helical peptides, suchas transportan and model amphipathic peptide (MAP), where lysine (Lys)is the main contributor to the positive charge, while the second classincludes arginine (Arg)-rich peptides, such as TAT and Antp orpenetratin.

The nucleic acids according to (e) include the sequences with the SEQ IDNOs: 12, 16, 8 and 20 as well those fragments thereof which comprise asequence encoding any of the fragments according to (d). Said nucleicacid may optionally comprise a sequence encoding a cell-penetratingpeptide.

Also embraced by the present invention are pharmaceutical compositionscomprising fragments of proteins orthologous or homologous to hNOXA1,hNOXO1, hp47phox/NOXO2 or hp67phox/NOXA2, whereby said fragments alignwith the fragments of hNOXA1, hNOXO1, hp47phox/NOXO2 or hp67phox/NOXA2recited under (d), as are pharmaceutical compositions comprising nucleicacids encoding these aligning fragments. It is understood that thesepharmaceutical compositions are considered equivalents of the abovedescribed embodiment directed to pharmaceutical compositions. Saidorthologous or homologous proteins include the respective murineproteins, i.e., proteins having an amino acid sequence set forth in anyone of SEQ ID NO: 13, 17, 9 or 21. The nucleic acids encoding the latterare set forth in SEQ ID NO: 14, 18, 10 and 22.

Two nucleotide or protein sequences can be aligned electronically usingsuitable computer programs known in the art. Such programs compriseBLAST (Altschul et al. (1990), J. Mol. Biol. 215, 403-410), variantsthereof such as WU-BLAST (Altschul & Gish (1996), Methods Enzymol. 266,460-480), FASTA (Pearson & Lipman (1988), Proc. Natl. Acad. Sci. USA 85,2444-2448) or implementations of the Smith-Waterman algorithm (SSEARCH,Smith & Waterman (1981), J. Mol. Biol. 147, 195-197). These programs, inaddition to providing a pairwise sequence alignment, also report thesequence identity level (usually in percent identity) and theprobability for the occurrence of the alignment by chance (P-value).Programs such as CLUSTALW (Higgins et al. (1994), Nucleic Acids Res. 22,4673-4680) can be used to align more than two sequences.

Furthermore embraced by the present invention are pharmaceuticalcompositions comprising (a) peptidomimetic compound(s) which has beenobtained by using any of the fragments according to (d) as a leadcompound.

Pharmaceutical compositions comprising a nucleic acid according to (e)and/or the above described equivalents thereof are also envisaged to beused for gene therapy. For this purpose, the nucleic acid may be part ofan expression, a gene transfer or gene targeting vector. Gene therapy,which is based on introducing therapeutic genes into cells by ex-vivo orin-vivo techniques is one of the most important applications of genetransfer. Transgenic mice expressing a neutralizing antibody directedagainst nerve growth factor have been generated using the“neuroantibody” technique; Capsoni, Proc. Natl. Acad. Sci. USA 97(2000), 6826-6831 and Biocca, Embo J. 9 (1990), 101-108. Suitablevectors, methods or gene-delivering systems for in-vitro or in-vivo genetherapy are described in the literature and are known to the personskilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996),534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256(1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res. 77 (1995), 1077-1086; Onodua, Blood 91 (1998), 30-36; Verzeletti,Hum. Gene Ther. 9 (1998), 2243-2251; Verma, Nature 389 (1997), 239-242;Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997),393-400; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO97/00957; U.S. Pat. No. 5,580,859; U.S. Pat. No. 5,589,466; U.S. Pat.No. 4,394,448 or Schaper, Current Opinion in Biotechnology 7 (1996),635-640, and references cited therein. The nucleic acid moleculesaccording to (e) may be designed for direct introduction or forintroduction via liposomes, viral vectors (e.g. adenoviral, retroviral),electroporation, ballistic (e.g. gene gun) or other delivery systemsinto the cell. Additionally, a baculoviral system can be used aseukaryotic expression system for the nucleic acid molecules of theinvention. The introduction and gene therapeutic approach should,preferably, lead to the expression of a fragment according to (d) of theinvention, whereby said expressed fragment is particularly useful in thetreatment, amelioration and/or prevention of hearing loss and/or phantomhearing.

Said antibody, which is monoclonal antibody, polyclonal antibody, singlechain antibody, or fragment thereof that specifically binds said peptideor polypeptide also including bispecific antibody, synthetic antibody,antibody fragment, such as Fab, a F(ab₂)′, Fv or scFv fragments etc., ora chemically modified derivative of any of these (all comprised by theterm “antibody”). Monoclonal antibodies can be prepared, for example, bythe techniques as originally described in Köhler and Milstein, Nature256 (1975), 495, and Galfré, Meth. Enzymol. 73 (1981), 3, which comprisethe fusion of mouse myeloma cells to spleen cells derived from immunizedmammals with modifications developed by the art. Furthermore, antibodiesor fragments thereof to the aforementioned peptides can be obtained byusing methods which are described, e.g., in Harlow and Lane “Antibodies,A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. Whenderivatives of said antibodies are obtained by the phage displaytechnique, surface plasmon resonance as employed in the BIAcore systemcan be used to increase the efficiency of phage antibodies which bind toan epitope of the peptide or polypeptide of the invention (Schier, HumanAntibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods183 (1995), 7-13). The production of chimeric antibodies is described,for example, in WO89/09622. A further source of antibodies to beutilized in accordance with the present invention are so-calledxenogenic antibodies. The general principle for the production ofxenogenic antibodies such as human antibodies in mice is described in,e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. Antibodiesto be employed in accordance with the invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook (1989), loc. cit.

The term “monoclonal” or “polyclonal antibody” (see Harlow and Lane,(1988), loc. cit.) also relates to derivatives of said antibodies whichretain or essentially retain their binding specificity. Whereasparticularly preferred embodiments of said derivatives are specifiedfurther herein below, other preferred derivatives of such antibodies arechimeric antibodies comprising, for example, a mouse or rat variableregion and a human constant region.

The term “scFv fragment” (single-chain Fv fragment) is well understoodin the art and preferred due to its small size and the possibility torecombinantly produce such fragments.

Preferably, the antibody, aptamer, fragment or derivative thereofaccording to the invention specifically binds the target protein,(poly)peptide or fragment or epitope thereof whose presence or absenceis to be monitored.

The term “specifically binds” in connection with the antibody used inaccordance with the present invention means that the antibody etc. doesnot or essentially does not cross-react with (poly)peptides of similarstructures. Cross-reactivity of a panel of antibodies etc. underinvestigation may be tested, for example, by assessing binding of saidpanel of antibodies etc. under conventional conditions (see, e.g.,Harlow and Lane, (1988), loc. cit.) to the (poly)peptide of interest aswell as to a number of more or less (structurally and/or functionally)closely related (poly)peptides. Only those antibodies that bind to the(poly)peptide/protein of interest but do not or do not essentially bindto any of the other (poly)peptides which are preferably expressed by thesame tissue as the (poly)peptide of interest, are considered specificfor the (poly)peptide/protein of interest and selected for furtherstudies in accordance with the method of the invention.

In a particularly preferred embodiment of the method of the invention,said antibody or antibody binding portion is or is derived from a humanantibody or a humanized antibody.

The term “humanized antibody” means, in accordance with the presentinvention, an antibody of non-human origin, where at least onecomplementarity determining region (CDR) in the variable regions such asthe CDR3 and preferably all 6 CDRs have been replaced by CDRs of anantibody of human origin having a desired specificity. Optionally, thenon-human constant region(s) of the antibody has/have been replaced by(a) constant region(s) of a human antibody. Methods for the productionof humanized antibodies are described in, e.g., ER-A1 0 239 400 andWO90/07861.

The term “aptamer” as used herein refers to DNA or RNA molecules thathave been selected from random pools based on their ability to bindother molecules. Aptamers have been selected which bind nucleic acid,proteins, small organic compounds, and even entire organisms. A databaseof aptamers is maintained at http://aptamer.icmb.utexas.edu/.

An antisense nucleic acid according to the invention is a nucleic acidmolecule complementary to a nucleic acid molecule encoding a proteinaccording to the main embodiment which may be used for the repression ofexpression of said protein. The construction of small interfering RNAs(siRNAs) (see, e.g. Zamore Nat Struct Biol 2001, 8(9):746-50 or TuschlT. CHEMBIOCHEM. 2001, 2:239-245) or of appropriate ribozymes (see, e.g.,EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specificallycleave the (pre)-mRNA of a gene comprising a nucleic acid encoding saidprotein are also suitable for the repression of expression. Thetechniques underlying said repression of expression are well known inthe art. Selection of appropriate target sites and correspondingribozymes can be done as described for example in Steinecke et al.(Methods in Cell Biology (1995) 50:449-460). Standard methods relatingto antisense technology have also been described (Melani et al., CancerRes. (1991) 51:2897-2901). Said nucleic acid molecules may be chemicallysynthesized or transcribed by an appropriate vector containing achimeric gene which allows for the transcription of said nucleic acidmolecule in the cell. Such nucleic acid molecules may further containribozyme sequences as described above.

Iodonium derivatives or, more specifically, aryliodonium compoundsinclude diphenylene iodonium (DPI, also referred to as iodoniumdiphenylor iodonium biphenyl), di-2-thienyliodonium (also referred to asiodonium thiophene) and phenoxaiodonium. These compounds act asarylating agents and directly and irreversibly inhibit NOX enzymes.

Apocynin (4-hydroxy-3-methoxy-acetophenone) is a methoxy-substitutedcatechol and exerts its effect on NOX enzymes through the inhibition ofsubunit assembly.

Also embraced by the present invention are pharmaceutical compositionscomprising (i) naphthoquinones such as plumbagin, acetylshikonin; (ii)inhibitors of HMG-CoA reductase including statins such as lovastatin,simvastatin, atorvastatin; (iii) gliotoxin; (iv) phenothiazines such asphenothiazine, trifluoperazine, and/or (v) a derivative of any one of(i) to (v).

Plumbagin is a naphtoquinone derived from Plumbago Zeylanica (Chitrak,an indian medicinal plant).

Gliotoxin is a metabolite of pathogenic fungi (Aspergillus and Candidaspp) and has been implicated in infectious pathways. It exhibitsimmunosupressive action and antitumor activity and inhibits activationprocess of NOX2 (Yoshida et al., 2000) and the assembly of the enzyme(Tsunawaki et al., 2004). It is available from Sigma. Statins areinhibitors of HMG-CoA. They decrease plasma cholesterol and block rac-1dependent activation of NADPH oxidases (Maack et al. 2003). Furthermore,they inhibit myristoylation of rac.

Trifluoperazine is an inhibitor of PKC/calmodulin and prevents theactivation of NADPH oxidases (Seifert and Scachtele, 1988, Holland etal., 2000).

The term derivative relates to compounds having the same core orbackbone structure while one or more of the substituents are modified,for example by replacing a methyl group with a trifluoromethyl group.These modifications are such that the biological/pharmacologicalactivity is not substantially altered. Said activity may be monitored bythe assays disclosed herein.

The present invention also relates to a pharmaceutical compositionconsisting of (a) ortho-methoxy-substituted catechols such as apocynin,acetosyringone, vanillin, vanillic acid, syringaldehyde, syringic acid;and (b) a pharmaceutically acceptable carrier, excipient or diluent.

Also provided by the present invention is a pharmaceutical compositioncomprising (a) an ototoxic agent; and (b) a compound selected from thegroup consisting of: (i) an antibody, aptamer, or a fragment orderivative thereof binding specifically the protein defined in claim 1;(ii) an antisense nucleic acid, an siRNA, or a ribozyme bindingspecifically a nucleic acid encoding said protein; (iii) a compoundcomprising the fragment of SEQ ID NO: 11 from position 202 to position212, the fragment of SEQ ID NO: 11 from position 402 to position 463,the fragment of SEQ ID NO: 15 from position 200 to position 210, thefragment of SEQ ID NO: 15 from position 457 to position 513, thefragment of SEQ ID NO: 7 from position 158 to position 217, the fragmentof SEQ ID NO: 7 from position 233 to position 289, the fragment of SEQID NO: 7 from position 321 to position 331, the fragment of SEQ ID NO:19 from position 156 to position 216, the fragment of SEQ ID NO: 19 fromposition 226 to position 286, the fragment of SEQ ID NO: 19 fromposition 360 to position 370, wherein said compound may furthermorecomprise a cell-penetrating peptide; (iv) a nucleic acid comprising asequence encoding any of the fragments according to (c), wherein saidnucleic acid may optionally comprise a sequence encoding acell-penetrating peptide; (v) aryliodonium compounds such as diphenyleneiodonium (DPI), di-2-thienyliodonium, phenoxaiodonium; (vi)naphthoquinones such as plumbagin, acetylshikonin; (vii) inhibitors ofHMG-CoA reductase including statins such as lovastatin, simvastatin,atorvastatin; (viii) gliotoxin; (ix) phenothiazines such asphenothiazine, trifluoperazine, and/or (x) a derivative of any one of(v) to (ix). Said ototoxic agent may be any agent detailed herein above.Preferably, said ototoxic agent is a medicament, wherein said medicamentcauses ototoxicity as a side effect. Therefore, and in view of thedisclosure of the mechanism of ototoxicity in this application, acombination therapy with a medicament with ototoxic side effect and aninhibitor of the protein defined in the main embodiment is provided.Also provided is the use of an ototoxic agent and of a compound asdefined in (b) above for the manufacture of pharmaceutical composition,wherein said compound as defined in (b) prevents, alleviates or curesthe ototoxic effect of said ototoxic agent.

In a preferred embodiment of said pharmaceutical composition, saidototoxic agent is an antibiotic.

In a more preferred embodiment of said pharmaceutical composition, saidototoxic agent is an aminoglycoside antibiotic, preferably gentamycin.This type of combination therapy is particularly envisaged for thoseregions or countries where aminoglycoside antibiotics such asgentamycin, owing to their low cost, are widely used.

The present invention also relates to the use of a modulator of theprotein defined in the main embodiment for the preparation of apharmaceutical composition for the treatment and/or prevention ofhearing loss and/or phantom hearing, wherein said modulator is selectedfrom the group consisting of (a) an antibody, aptamer, or a fragment orderivative thereof binding specifically said protein; (b) an antisensenucleic acid, an siRNA, or a ribozyme binding specifically a nucleicacid encoding said protein; (c) a known modulator of NOX3 and/or NADPHoxidases and/or electron transport proteins; (d) a compound comprisingthe fragment of SEQ ID NO: 11 from position 202 to position 212, thefragment of SEQ ID NO: 11 from position 402 to position 463, thefragment of SEQ ID NO: 15 from position 200 to position 210, thefragment of SEQ ID NO: 15 from position 457 to position 513, thefragment of SEQ ID NO: 7 from position 158 to position 217, the fragmentof SEQ ID NO: 7 from position 233 to position 289, the fragment of SEQID NO: 7 from position 321 to position 331, the fragment of SEQ ID NO:19 from position 156 to position 216, the fragment of SEQ ID NO: 19 fromposition 226 to position 286, the fragment of SEQ ID NO: 19 fromposition 360 to position 370; (e) a nucleic acid comprising a sequenceencoding any of the fragments according to (d); and (f) a modulatoridentified by the method of any one of claims 1 to 13. The fragmentsaccording to (d) are regions of the sequences of the respective SEQ IDNOs known or expected to be involved in subunit association. Saidcompounds according to (d) may furthermore comprise a cell-penetratingpeptide. The term “cell-penetrating peptide” is defined herein above.

The nucleic acids according to (e) include the sequences with the SEQ IDNOs: 12, 16, 8 and 20 as well those fragments thereof which comprise asequence encoding any of the fragments according to (d). Said nucleicacid may optionally comprise a sequence encoding a cell-penetratingpeptide.

Also embraced by the present invention are uses of fragments of proteinsorthologous or homologous to hNOXA1, hNOXO1, hp47phox/NOXO2 orhp67phox/NOXA2, whereby said fragments align with the fragments ofhNOXA1, hNOXO1, hp47phox/NOXO2 or hp67phox/NOXA2 recited under (d), asare uses of nucleic acids encoding these aligning fragments. It isunderstood that these uses are considered equivalents of the abovedescribed embodiment. Said orthologous or homologous proteins includethe respective murine proteins, i.e., proteins having an amino acidsequence set forth in any one of SEQ ID NO: 13, 17, 9 or 21. The nucleicacids encoding the latter are set forth in SEQ ID NO: 14, 18, 10 and 22.

Furthermore embraced by the present invention are uses of (a)peptidomimetic compound(s) which has been obtained by using any of thefragments according to (d) as a lead compound.

Uses of a nucleic acid according to (e) and/or of the above describedequivalents thereof are also envisaged for gene therapy.

The present invention also relates to the use of a cisplatin and/orhydrogen hexachloroplatinate for the preparation of a pharmaceuticalcomposition for the treatment and/or prevention of tinnitus. Cisplatinand hydrogen hexachloroplatinate are activators of the protein definedthe main embodiment. Surprisingly, in many incidences of tinnitus apositive response to a treatment with compounds known to induceoxidative stress in the inner ear is observed.

Also provided is a method of diagnosing hearing loss and/or phantomhearing and/or an individual's disposition or risk to develop said lossand/or said phantom hearing, comprising the steps of: (a) determining(a) polymorphism(s) in a NOX3 gene or cDNA and/or in a gene or cDNAencoding an NADPH oxidase subunit in a sample obtained from saidindividual; and (b) associating said polymorphism(s) with a diseasestate or disposition state. Preferably, said sample is a blood sample.Preferably, said NOX3 gene comprises or consists of the sequence setforth in SEQ ID NO: 23 or 24. Preferably said NOX3 cDNA (or equivalentlymRNA) comprises or consists of the sequence set forth in SEQ ID NO: 2, 4or 6. Preferably said cDNA encoding an NADPH oxidase subunit comprisesor consists of the sequence set forth in any one of SEQ ID NOs: 8, 10,12, 14, 16, 18, 20 or 22.

The term “polymorphism”, or “nucleotide polymorphism” refers to theoccurrence of one or more different nucleotides or bases at a givenlocation on a chromosome. Usually, polymorphisms are distinguished frommutations based on their prevalence. Sometimes a threshold of 1%prevalence in a population of individuals is considered for separatingpolymorphisms (more frequent) from mutations (less frequent). A singlenucleotide polymorphism (SNP) is a polymorphism of a single nucleotideor base. The SNP database maintained at NCBI(http://www.ncbi.nlm.nih.gov/SNP/) divides SNPs into SNPs in theproximity of a known locus and such that are 5′ further away than 2 kbfrom the most 5′ feature of a gene and 3′ further away than 500 basesfrom the most 3′ feature of a gene. SNPs in the proximity of a knownlocus are further divided into SNPs occurring at an mRNA location andsuch that do not. SNPs occurring at an mRNA location comprise coding andnon-coding SNPs.

It is understood that the term “polymorphism(s) in a NOX3 gene and/or ina gene encoding an NADPH oxidase subunit” embraces polymorphisms inexons, introns and regulatory regions such as promoters. Polymorphismsin exons may be determined or analysed using genomic DNA or cDNA (orequivalently mRNA). Polymorphisms in introns or regulatory regions suchas promoters may be determined or analysed using cDNA (or equivalentlymRNA).

Said associating of polymorphism(s) with a disease state or dispositionstate refers to classifying of individuals and patients. The term“classifying” refers to the assignment of individuals or patients to twoor more groups or classes. In other words, individuals, previouslyunclassified, get labelled by their respective class. The assigned classlabel may refer to parameters used for classification, e.g.polymorphisms, or may refer to parameters not used for classificationbecause their values are not known beforehand, e.g. fast or slowresponse to therapy. In the first case, class discovery methods, e.g.clustering may be applied, whereas in the second case predictiveclassification methods are used. Classification may be done manually bya trained person or by a computer program provided with the values ofthe parameters used for class distinction. Patients have to giveinformed consent. Data have to be handled and kept secret in accordancewith national laws.

The present invention also provides the use of a compound binding to theprotein defined in the main embodiment or to a NADPH oxidase subunit forthe preparation of a diagnostic composition for the diagnosis of hearingloss and/or phantom hearing and/or an individual's disposition or riskto develop said loss and/or said phantom hearing, wherein said compoundis selected from the group consisting of (a) an antibody, aptamer, or afragment or derivative thereof binding specifically said protein; and(b) a known modulator of NOX3 and/or NADPH oxidases and/or electrontransport proteins.

In a preferred embodiment of the use according to the invention, saidknown modulator is selected from the group consisting of iodoniumderivatives, substituted catechols such as apocynin, platinumderivatives and palladium derivatives.

Known modulators to be used for the preparation of a pharmaceuticalcomposition according to the invention are selected from the groupconsisting of (i) aryliodonium compounds such as diphenylene iodonium(DPI), di-2-thienyliodonium, phenoxaiodonium; (ii)ortho-methoxy-substituted catechols such as apocynin, acetosyringone,vanillin, vanillic acid, syringaldehyde, syringic acid; (iii)naphthoquinones such as plumbagin, acetylshikonin; (iv) inhibitors ofHMG-CoA reductase including statins such as lovastatin, simvastatin,atorvastatin; (v) gliotoxin; (vi) phenothiazines such as phenothiazine,trifluoperazine; and (vii) a derivative of any one of (i) to (vi). Saidknown modulators act as inhibitors of the protein defined in the mainembodiment.

Known modulators to be used for the preparation of a diagnosticcomposition according to the invention are selected from the knownmodulators to be used for the preparation of a pharmaceuticalcomposition and cisplatin and hexachloroplatinate as well as derivativesthereof. Cisplatin and hexachloroplatinate bind and activate the proteindefined in the main embodiment and are therefore specifically envisagedfor the manufacture of a diagnostic composition.

Cisplatin, as demonstrated by the inventors, is a preferred platinumderivative which modulates NOX3 activity. The platinum derivativehydrogen hexachloroplatinate and palladium derivatives are known tomodulate the activity of NOX2 (phagocyte NADPH oxidase). In both cases,there are indications that modulation is a direct effect on the NOXenzymes.

Also envisaged is the use of a compound binding to a nucleic acidencoding the protein defined in the main embodiment or an NADPH oxidasesubunit for the preparation of a diagnostic composition for thediagnosis of hearing loss and/or phantom hearing and/or an individual'sdisposition or risk to develop said loss and/or said phantom hearing,wherein said compound is a nucleic acid complementary to said nucleicacid and at least 15 nucleotides in length. This embodiment is directedto oligonucleotide probes for the detection of genomic DNA or mRNA. Withregard to genomic DNA, also the detection and distinction ofpolymorphisms is envisaged.

Preferably, said compound is detectably labelled.

More preferred, said diagnosis to be performed involves imaging of thehuman or animal body.

In a preferred embodiment of the method or the use of the invention,said animal is a rodent. More preferred, said rodent is mouse or rat.

In a preferred embodiment of the method or the use of the presentinvention, said modulator is an inhibitor.

The term “inhibitor” designates a compound lowering the activity of atarget molecule, preferably by performing one or more of the followingeffects: (i) the transcription of the gene encoding the protein to beinhibited is lowered, (ii) the translation of the mRNA encoding theprotein to be inhibited is lowered, (iii) the protein performs itsbiochemical function with lowered efficiency in presence of theinhibitor, and (iv) the protein performs its cellular function withlowered efficiency in presence of the inhibitor.

Compounds falling in class (i) include compounds interfering with thetranscriptional machinery and/or its interaction with the promoter ofsaid gene and/or with expression control elements remote from thepromoter such as enhancers. Compounds of class (ii) comprise antisenseconstructs and constructs for performing RNA interference well known inthe art (see, e.g. Zamore (2001) or Tuschl (2001)). Compounds of class(iii) interfere with molecular function of the protein to be inhibited,in case of an NADPH oxidase with its enzymatic activity and/or itscapability to associate with NADPH oxidase subunits. Accordingly, activesite binding compounds, in particular compounds capable of binding tothe active site of any NADPH oxidase, are envisaged, as are compoundsinterfering with the association of NADPH oxidase with said subunits.More preferred are compounds specifically binding to an active site ofNADPH oxidase. Also envisaged are compounds binding to or blockingsubstrate binding sites of NADPH oxidase. Class (iv) includes compoundswhich do not necessarily directly bind to NADPH oxidase, but stillinterfere with NADPH oxidase activity, for example by binding to and/orinhibiting the function or inhibiting expression of members of a pathwaywhich comprises NADPH oxidase. These members may be either upstream ordownstream of NADPH oxidase within said pathway.

In a preferred embodiment, the inhibitor is a low molecular weightcompound. Low molecular weight compounds are compounds of natural originor chemically synthesized compounds, preferably with a molecular weightbetween 100 and 1000, more preferred between 200 and 750, and even morepreferred between 300 and 600.

The efficiency of the inhibitor can be quantitized by comparing thelevel of activity in the presence of the inhibitor to that in theabsence of the inhibitor. For example, as an activity measure may beused: the change in amount of mRNA formed, the change in amount ofprotein formed, the change in amount of substrate converted or productformed, and/or the change in the cellular phenotype or in the phenotypeof an organism.

In a preferred embodiment, the level of activity is less than 90%, morepreferred less than 80%, 70%, 60% or 50% of the activity in absence ofthe inhibitor. Yet more preferred are inhibitors lowering the level downto less than 25%, less than 10%, less than 5% or less than 1% of theactivity in absence of the inhibitor.

The present invention also relates to a nucleic acid (i) comprising orconsisting of the sequence of SEQ ID NO: 6, or (ii) encoding a proteincomprising or consisting of the sequence of SEQ ID NO: 5, or (iii)encoding a fragment of the protein according to (ii), wherein saidfragment exhibits NADPH oxidase activity, or (iv) encoding a proteinhaving a sequence at least 95% identical with the protein according to(ii) or with the fragment according to (iii) and exhibiting NADPHoxidase activity.

Preferably, said protein having at least 95% sequence identity with theprotein according to (ii) or with the fragment according to (iii), has98% or 99% identity with said protein or fragment.

An alternative embodiment of the invention relates to a vectorcomprising the above defined nucleic acid.

The vector of the present invention may be, e.g., a plasmid, cosmid,virus, bacteriophage or another vector used e.g. conventionally ingenetic engineering, and may comprise further genes such as marker geneswhich allow for the selection of said vector in a suitable host cell andunder suitable conditions.

Furthermore, the vector of the present invention may, in addition to thenucleic acids of the invention, comprise expression control elements,allowing proper expression of the coding regions in suitable hosts. Suchcontrol elements are known to the artisan and may include a promoter, asplice cassette, translation initiation codon, translation and insertionsite for introducing an insert into the vector. Preferably, the nucleicacid of the invention is operably linked to said expression controlsequences allowing expression in eukaryotic or prokaryotic cells.

Many suitable vectors are known to those skilled in molecular biology,the choice of which would depend on the function desired and includeplasmids, cosmids, viruses, bacteriophages and other vectors usedconventionally in genetic engineering. Methods which are well known tothose skilled in the art can be used to construct various plasmids andvectors; see, for example, the techniques described in Sambrook (1989),loc. cit., and Ausubel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y. (1989), (1994).Alternatively, the nucleic acids and vectors of the invention can bereconstituted into liposomes for delivery to target cells. According tothe invention relevant sequences can be transferred into expressionvectors where expression of a particular (poly)peptide/protein isrequired. Typical cloning vectors include pBscpt sk, pGEM, pUC9, pBR322and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1,pOP13CAT.

Furthermore, a protein encoded by said nucleic acid is provided.

The present invention furthermore relates to host containing anaforementioned vector or an aforementioned nucleic acid, or anaforementioned protein. Said host may be produced by introducing saidvector or nucleic acid into a host cell which upon its presence in thecell mediates the expression of a protein encoded by the nucleic acid ofthe invention or comprising a nucleic acid or a vector according to theinvention wherein the nucleic acid and/or the encoded(poly)peptide/protein is foreign to the host cell.

By “foreign” it is meant that the nucleic acid and/or the encoded(poly)peptide/protein is either heterologous with respect to the host,this means derived from a cell or organism with a different genomicbackground, or is homologous with respect to the host but located in adifferent genomic environment than the naturally occurring counterpartof said nucleic acid. This means that, if the nucleic acid is homologouswith respect to the host, it is not located in its natural location inthe genome of said host, in particular it is surrounded by differentgenes. In this case the nucleic acid may be either under the control ofits own promoter or under the control of a heterologous promoter. Thevector or nucleic acid according to the invention which is present inthe host may either be integrated into the genome of the host or it maybe maintained in some form extrachromosomally. In this respect, it isalso to be understood that the nucleic acid of the invention can be usedto restore or create a mutant gene via homologous recombination.

The host can be any prokaryote or eukaryotic cell, such as a bacteria,an insect, fungal, plant or animal cell.

The term “prokaryote” is meant to include all bacteria which can betransformed or transfected with a DNA or RNA molecules for theexpression of a protein of the invention. Prokaryotic hosts may includegram negative as well as gram positive bacteria such as, for example, E.coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Theterm “eukaryotic” is meant to include yeast cells, cells of higherplant, insect cells and preferably mammalian cells. Depending upon thehost employed in a recombinant production procedure, the protein encodedby the nucleic acid of the present invention may be glycosylated or maybe non-glycosylated. A nucleic acid of the invention can be used totransform or transfect the host using any of the techniques commonlyknown to those of ordinary skill in the art. Furthermore, methods forpreparing fused, operably linked genes and expressing them in, e.g.,mammalian cells and bacteria are well-known in the art (Sambrook (1989),loc. cit.).

Preferably, said host is a cell. More preferred, the host is a humancell or human cell line.

Alternatively, said host is a transgenic non-human animal.

A method for the production of a transgenic non-human animal, forexample transgenic mouse, comprises introduction of a nucleic acid orvector according to the invention into a germ cell, an embryonic cell,stem cell or an egg or a cell derived therefrom. The non-human animalcan be used in accordance with a screening method of the inventiondescribed herein. Production of transgenic embryos and screening ofthose can be performed, e.g., as described by A. L. Joyner Ed., GeneTargeting, A Practical Approach (1993), Oxford University Press. The DNAof the embryonal membranes of embryos can be analyzed using, e.g.,Southern blots with an appropriate probe. A general method for makingtransgenic non-human animals is described in the art, see for example WO94/24274. For making transgenic non-human organisms (which includehomologously targeted non-human animals), embryonal stem cells (EScells) are preferred. Murine ES cells, such as AB-1 line grown onmitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley,Cell 62: 1073-1085 (1990)) essentially as described (Robertson, E. J.(1987) in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112) may beused for homologous gene targeting. Other suitable ES lines include, butare not limited to, the E14 line (Hooper et al., Nature 326: 292-295(1987)), the D3 line (Doetschman et al., J. Embryol. Exp. Morph. 87:27-45 (1985)), the CCE line (Robertson et al., Nature 323: 445-448(1986)), the AK-7 line (Zhuang et al., Cell 77: 875-884 (1994) which isincorporated by reference herein). The success of generating a mouseline from ES cells bearing a specific targeted mutation depends on thepluripotence of the ES cells (i.e., their ability, once injected into ahost developing embryo, such as a blastocyst or morula, to participatein embryogenesis and contribute to the germ cells of the resultinganimal). The blastocysts containing the injected ES cells are allowed todevelop in the uteri of pseudopregnant non-human females and are born aschimeric mice. The resultant transgenic mice are chimeric for cellshaving either the recombinase or reporter loci and are backcrossed andscreened for the presence of the correctly targeted transgene(s) by PCRor Southern blot analysis on tail biopsy DNA of offspring so as toidentify transgenic mice heterozygous for either the recombinase orreporter locus/loci.

Methods for producing transgenic flies, such as Drosophila melanogasterare also described in the art, see for example U.S. Pat. No. 4,670,388,Brand & Perrimon, Development (1993) 118: 401-415; and Phelps & Brand,Methods (April 1998) 14: 367-379.

Transgenic worms such as C. elegans can be generated as described inMello, et al., (1991) Efficient gene transfer in C. elegans:extrachromosomal maintenance and integration of transforming sequences.Embo J 10, 3959-70, Plasterk, (1995) Reverse genetics: from genesequence to mutant worm. Methods Cell Biol 48, 59-80.

The invention also relates to transgenic non-human animals such astransgenic mouse, rats, hamsters, dogs, monkeys, rabbits, pigs, C.elegans and fish such as Torpedo fish comprising a nucleic acidaccording to the invention.

Also provided is an antibody or aptamer, or fragment or derivativethereof binding specifically to the protein encoded by said nucleic acidas is an antisense nucleic acid, an siRNA, or a ribozyme bindingspecifically said nucleic acid.

The Figures show:

FIG. 1: Tissue distribution of NOX3 mRNA. A) NOX3 mRNA expression wasevaluated in 12 rat tissues by RT-PCR (upper panel); GAPDH mRNA was usedas a reference transcript (lower panel). “No cDNA” represents negativecontrol PCR devoid of added cDNA. The first lane of both panels showsDNA size markers. B) Quantification of NOX3 RNA in 14 mouse tissuesusing real time PCR. NOX3 mRNA expression is shown relative to 18S rRNAexpression. The amounts of NOX3 and 18S PCR products were measured usingSYBR Green.

FIG. 2: PCR detection of cDNAs encoding NOX activator and regulatorsubunits in the inner ear. A, RT-PCR amplification of NOXA1, NOXO1, andthe reference GAPDH cDNA from the indicated rat tissues. B, RT-PCRamplification of p67^(phox) and p47^(phox) cDNA from the indicated rattissues. The first lane of each panel shows DNA size markers.

FIG. 3: Expression of NOX3 mRNA in specific regions of cochlea. Theindicated regions of the rat inner ear were obtained by microdissectionand NOX3 (upper panel) and GAPDH (lower panel) expression were assessedby RT-PCR. “+” symbols represent reverse transcribed (RT positive)samples; “−” symbols represent not reverse transcribed (RT negative)samples. P0, P3, and P4 indicate the postnatal days when samples weretaken. Positive control inner ear sample was isolated from adult rat.

FIG. 4: Localization of NOX3 mRNA in inner ear by in situ hybridization.Mouse inner ear sections hybridized with digoxigenin-labeled antisense(A, C, and E) and sense (B, D, and F) probes of NOX3, shown at ×20 (A,B) and ×40 (C-F) magnifications. A, The antisense probe hybridized withthe RNA of spiral ganglion neurons. B, The sense probe yielded only aweak, uniform signal and no labeling of spiral ganglion neurons. C,Hybridization of antisense NOX3 probe with the organ of Corti labeledthe sensory epithelium. D, Hybridization of sense NOX3 probe with organof Corti did not yield specific signals. E, Antisense NOX3 probehybridized with the sensory epithelial cell layer of the saccule. F,Only a week uniform signal was observed with the sense NOX3 probe.

FIG. 5: NOX3-dependent superoxide production in the absence of other NOXsubunits. HEK293 cells were transfected with either pcDNA3.1 vector orNOX3, and superoxide generation was measured as cytochrome C reduction(upper panel) or as luminol-amplified chemiluminescence (lower panel) inthe presence or absence of 100 nM PMA, as indicated. Upper panel showsthe result of a single experiment representative of three independentstudies. Lower panel shows statistical analysis of peak superoxideproduction. Chemiluminescence signals were measured with relative lightunits (RLU) and normalized to 1 second and 150,000 cells.

FIG. 6: Subunit regulation of NOX3 activity. A, B, and C, HEK293 cellswere transfected with different combinations of NOX3, NOXO1, NOXA1,p47^(phox), and p67^(phox), as indicated. Superoxide generation wasmeasured as SOD sensitive cytochrome C reduction (lines and symbols) oras luminol-amplified chemiluminescence (bar graphs) in the presence orabsence of PMA (100 nM), as indicated. Lines and symbols show typicalexperiments, representative of at least three independent studies. Bargraphs show statistical analysis of peak superoxide production.Chemiluminescence signals were measured with relative light units (RLU)and normalized to 1 second and 150,000 cells.

FIG. 7: Cisplatin enhances NOX3-dependent superoxide production.Superoxide production of transfected HEK293 cells were measured eitheras luminol-amplified chemiluminescence (B, D, E and F) or with aluminol-based superoxide detection kit, Diogenes (A and C). Cells werepre-incubated in the presence or absence of 20 μM cisplatin for 12 hours(A-E). A, HEK293 cells were transfected with NOX3 or control vector(pcDNA3.1) and incubated with or without cisplatin before superoxidemeasurement. 100 nM PMA and 5 μM DPI were added as indicated. Tracesrepresent a typical experiment, representative of three independentstudies. B, HEK293 cells stably expressing NOX3/NOXA1/NOXO1 werepre-incubated with or without cisplatin before superoxide measurement. 5μM DPI was added as indicated. Traces show a typical experiment,representative of eight independent studies. C, Statistical analysis ofpeak superoxide production of NOX3 transfected HEK293 cells, aftercisplatin- or control treatment, in the presence or absence of 100 nMPMA. D, Statistical analysis of peak superoxide production of HEK293cells transfected with the indicated constructs and pre-incubated withor without cisplatin. The measurements were carried out in the absenceor presence of 100 nM PMA, as indicated. E, Superoxide production of aHEK293 cell clone stably transfected with NOX3/NOXO1/NOXA1 afterincubation with various concentrations of cisplatin for 12 hours. F,Superoxide production of a HEK293 cell clone stably transfected withNOX3/NOXO1/NOXA1 after incubation in the presence or absence of 20 μMcisplatin for the indicated periods of time.

The following examples illustrate the invention but should not beconstrued as being limiting.

EXAMPLE 1 Cloning of Mouse and Rat NOX3 cDNA Experimental Procedures

The first and the last exons of mouse and rat NOX3 genes were identifiedbased on their homology with the human NOX3 gene using the EnsemblGenome Browser (www.ensembl.org). Inner ear samples of mouse (strainC57Bl6) and rat (strain Sprague-Dawley) were isolated and total RNA waspurified as described [28]. Primers were designed and used to amplifythe full length of coding sequences (mouse NOX3 forward 5′-atg ccg gtgtgc tgg att ctg aac-3′ and reverse 5′-cta gaa gtt ttc ctt gtt gta atagaa-3′, rat NOX3 forward 5′-gtg ttg gta gta aga gaa gtg tca tg-3′ andreverse 5′-c tag aag ttt tcc ttg ttg taa tag-3′) with Taq DNA polymerase(Qiagen) under standard conditions. PCR products were subcloned intopcDNA3.1 vector (Invitrogen) and verified by sequencing.

Results.

So far, NOX3 mRNA has only been detected in human embryonic kidney, butexpression levels were very low [22, 30] and hence the physiologicalrelevance questionable. We reasoned that the physiologically relevantlocalization of NOX3 might have been missed because previous studies hadrestricted their analysis to commercially available human RNA sources.To overcome these limitations, we decided to work in mouse and rat andto prepare RNA from tissues that had not been investigated so far. Ashitherto only the human NOX3 sequence was known, we identified mouse andrat NOX3 genes by searching genomic DNA databases and designed—based onthese results—mouse and rat NOX3 PCR primers.

We then prepared RNA from a variety of mouse and rat tissues, includingbone (femur, skull, shoulder blade), cartilage (joints of ribs, outerear), and inner ear and analyzed them for NOX3 expression by RT-PCR. Asshown on FIG. 1A, high levels of NOX3 transcript were detected only inthe rat inner ear sample (despite its relatively low mRNA contentdemonstrated by the low amount of GAPDH PCR product). Using primer pairsdesigned from the first and the last exons of the mouse and rat NOX3gene, respectively, we amplified whole length mouse and rat NOX3 codingsequences from inner ear samples. The predicted amino acid sequences ofboth mouse and rat NOX3 showed 81% sequence identity with the humansequence and 93.5% identity with each other.

EXAMPLE 2 Tissue Distribution of NOX3 Experimental Procedures

Total RNA was isolated from different organs of rat and mouse and fromspecific regions of the rat inner ear using the TRIzol reagent. With theexception of RNA purified from parts of the inner ear, samples wereDNase treated, then further purified with RNeasy kit (Qiagen). 2 μgtotal RNA from each tissue was reverse transcribed using Superscriptreverse transcriptase (Life Technologies, Inc.). PCR was carried outwith Taq DNA polymerase using the following primers: mouse NOX3 forward5′-gtg ata aca ggc tta aag cag aag gc-3′, reverse 5′-cca ctt tcc cct acttga ctt tag-3′; rat NOX3 forward 5′-gcg tgt gct gta gag gac cgt ggag-3′, reverse 5′-gag cct gtc cct ctg ctc caa atg c-3′; mouse GAPDHforward 5′-ggg tgt gaa cca cga gaa at-3′, reverse 5′-gtc atg agc cct tccaca at-3′; rat GAPDH forward 5′-cgg tgt caa cgg att tgg ccg tat t-3′,reverse 5′-act gtg gtc atg agc cct tcc acg a-3′; rat NOXO1 forward5′-acc caa acc tct gga tct gga gcc c-3′, reverse 5′-gga tgg cac tca tacagg ggc gag t-3′; rat NOXA1 forward 5′-tac tgg ccg tag cac gcg aag actg-3′, reverse 5′-gga cct ccc agg ctt gca gtt tga a-3′; rat p47^(phox)forward 5′-gca gga cct gtc gga gaa ggt ggt c-3′, reverse 5′-tct gtc gctggg cct ggg tta tct c-3′; rat p67^(phox) forward 5′-aag cag aag agc agttag cat tgg c-3′, reverse 5′-gga gtg cct tcc aaa ttc ttg gct g-3′.Standard PCR conditions were used, and the number of PCR cycles was 30(FIGS. 1 and 2) or 28 (FIG. 3) for the amplification of GAPDH and 35 forall other amplifications.

Quantitative PCR was carried out using ABI Prism 7900HT SequenceDetection System with standard temperature protocol and 2×SYBR Green PCRMaster Mix reagent (Applied Biosystems, Worrington, UK) in 25 μl volume,in triplicates. 300 nM of the following primer pairs were used for thereactions: mouse 18S forward 5′-aca tcc aag gaa ggc agc ag-3′ andreverse 5′-ttt tcg tca cta cct ccc cg-3′; mouse NOX3 forward 5′-cga cgaatt caa gca gat tgc-3′, and reverse 5′-aag agt ctt tga cat ggc tttgg-3′. All amplifications were carried out in a MicroAmp optical 96-wellreaction plate with optical adhesive covers (PE Applied Biosystems). Theaccumulation of PCR products was detected by monitoring the increase influorescence of the reporter dye.

Results.

NOX3 is Predominantly Expressed in the Inner Ear—

Based on the cDNA sequence of mouse NOX3, we designed primers for realtime PCR to study quantitative expression of NOX3 RNA in different mousetissues. 18S RNA was used as a reference gene. The results of real-timePCR demonstrated that NOX3 was predominantly expressed in the inner ear(FIG. 1B). Low amounts of NOX3 RNA could also be detected in skull,brain, and embryonic kidney. However, inner ear contained 50-fold of theNOX3 content of skull and 870-fold of the one of embryonic kidney (FIG.1B).

Expression of Cytoplasmic NOX Subunits in the Inner Ear—

NOX1 and gp91^(phox)/NOX2 require cytoplasmic organizer subunits (NOXO1,p47^(phox)) and activator subunits (NOXA1, p67^(phox)) to form afunctional enzyme. As NOX3 shows a high degree of homology with NOX1 andgp91^(phox)/NOX2 [31], we considered that it might also be asubunit-dependent enzyme and therefore investigated expression ofcytoplasmic NOX subunits in the inner ear. RT-PCR analysis (using 35 PCRcycles) showed that mRNA of the activator subunit NOXA1, as well as mRNAof the organizer subunit p47^(phox) was expressed in the inner ear (FIG.2). mRNA of the activator subunit, p67^(phox), and the organizersubunit, NOXO1, could be detected only at very high cycle numbers (40PCR cycles; data not shown). Since p47^(phox) mRNA is expressed inphagocytic cells, its detection might be due to blood cellcontamination. In contrast, NOXA1 is not expressed in blood cells [24]nor in tissues neighboring the inner ear (FIG. 2A); thus, it is mostlikely expressed within cells of the inner ear.

Expression of NOX3 in Different Parts of the Cochlea—

In order to identify regions of the inner ear that express NOX3, weisolated distinct parts of rat cochlea such as organ of Corti, striavascularis, and spiral ganglia from newborn rats (postnatal day 1 to 4)as described previously [32]. As a control tissue, we used dorsal rootganglia. Total RNA was extracted from these tissues and tested for NOX3and GAPDH housekeeping gene expression by RT-PCR. Results showed thatNOX3 is expressed in spiral ganglia and in the organ of Corti, whilestria vascularis and dorsal root ganglia were devoid of NOX3 mRNA (FIG.3). Our experiments demonstrated that i) NOX3 is expressed only inselected structures of the cochlea (i.e. organ of Corti and spiralganglia), and ii) its expression is not a general property of theperipheral nervous system (i.e. it was absent from dorsal root ganglia).

EXAMPLE 3 In Situ Hybridization Experimental Procedures

For in situ hybridization experiments digoxigenin-labelled antisense andsense (negative control) cRNA probes (nucleotides 560-849 of mNOX3) weregenerated and used as described previously [19] on decalcified, 7 μmthick inner ear sections.

Results.

To further define the site of NOX3 expression, we performed in situhybridization of adult mouse inner ear sections. The antisense NOX3probe labeled spiral ganglion neurons (FIG. 4A) and cells of the organof Corti (FIG. 4C). The cellular structures within the organ of Cortiwere not sufficiently well preserved to identify NOX3-expressing cellsmore precisely. The sense probe gave only a weak, uniform backgroundsignal demonstrating the specificity of the antisense hybridization(FIGS. 4 B and D). Specific labeling for NOX3 was also observed in thevestibular system, namely in the sensory epithelial cell layer of thesaccule (FIG. 4 E, F).

EXAMPLE 4 Measurement of Reactive Oxygen Species Experimental Procedures

Cell Culture and Transfection—

HEK293 were maintained in Dulbecco's Modified Eagle's Medium/Ham'sNutrient Mixture F12 that was supplemented with 10% fetal calf serum,penicillin (100 units/ml), streptomycin (100 μg/ml), and 4 mmol/literL-glutamine. NOX3-, NOXO1-, NOXA1-, p47^(phox), and p67^(phox) cDNAswere subcloned into pcDNA3.1 (Invitrogen, Groningen, Netherlands) andtransfected into HEK293 cells with the Effectene transfection system(Qiagen). To obtain stable clones, NOX3, NOXO1, NOXA1-transfected HEK293cells were selected with 400 μg/ml G418 starting on the 2nd day afterthe transfection. After 14 days of selection, 24 surviving clones weretested for superoxide production. The positive clones were verified toexpress NOX3-, NOXO1-, and NOXA1 RNA by RT-RCR.

ROS generation was measured by the peroxidase-dependentluminol-amplified chemiluminescence technique (referred to asluminol-amplified chemiluminescence) in 96 well microplates usingLuminometer Wallac 1420 Multilabel Counter (PerkinElmer Life Sciences).Measurements were performed in Hanks' balanced salt solutionsupplemented with 1 mg/ml D-glucose, 1 unit/ml horseradish peroxidase,and 250 μM luminol. In some experiments, phorbol ester (PMA) was addedduring the measurements to 100 nM final concentration. When the effectof cisplatin or 5-Fluorouracil (5-FU) was investigated, these compoundswere pre-incubated with the cells for the indicated time andconcentration in cell culture medium. Before ROS measurements, the cellculture medium was exchanged with the assay solution andchemiluminescence or absorption (see below) was measured at 37° C. Aftermeasurements cells were counted, and the results were normalized to150,000 cells. Extracellular superoxide production was measured in96-well microplates at 550 nm as the SOD-sensitive reduction of 100 μMferricytochrome C (referred to as cytochrome C reduction technique). TheO⁻ ₂ production was calculated using an absorption coefficient of 21.1mM⁻¹ cm⁻¹ and normalized to 10⁷ cells [29].

Results.

NOX3-Dependent Superoxide Generation in the Absence of Subunits—

To investigate its molecular function, we transiently expressed NOX3 inHEK293 cells, which do not show endogenous expression of the enzyme.Superoxide production was measured with cytochrome C reduction techniqueand with luminol-amplified chemiluminescence. Using either technique,NOX3-transfected cells generated low amounts of superoxide, but only inthe presence of a protein kinase C activator (phorbol ester, PMA) (FIG.5). Since both NOX1 and gp91^(phox)/NOX2 have an obligatory subunitrequirement, the stimulus-dependent and subunit-independent activity ofNOX3 is a unique and distinguishing feature of this NOX isoform.

Regulation of NOX3 by the Organizer and Activator Subunits of NOX1 andgp91^(phox)/NOX2—

Since expression of NOX regulator and activator subunits was detected inthe inner ear (see above, FIG. 2), we reasoned that they might influenceNOX3 activity. Thus, we investigated superoxide generation by NOX3 uponco-transfection with cytoplasmic subunits. In the first series ofexperiments, NOX3 was co-transfected with the cytosolic subunits of thephagocyte NADPH oxidase, p67^(phox) and p47^(phox). In thesetransfectants, the NOX3-dependent superoxide generation was markedlyincreased, even without an added stimulus (FIG. 6A). The addition ofPMA, however, led to a strong enhancement of NOX3 activity (FIG. 6A).HEK293 cells, transfected with p47^(phox) and p67^(phox) but devoid ofNOX3, did not produce any superoxide (not shown). Interestinglyp67^(phox) alone, in the absence of p47^(phox), was sufficient to doublethe PMA-induced superoxide generation of NOX3, while p47^(phox) in theabsence of p67^(phox), did not modify NOX3 activity (compare FIG. 5 withFIG. 6A). Next it was investigated whether NOX3 could be regulated bythe NOXO1 and NOXA1 subunits, which are associated with NOX1 in thecolon. Co-transfection of NOX3 with NOXO1 and NOXA1 resulted in amassive increase of superoxide production (FIG. 6B). TheNOXO1/NOXA1-enhanced superoxide generation was insensitive to PMA (FIG.6B). The co-expression of NOXA1 with NOX3, in the absence of NOXO1, hadan enhancing effect on PMA-stimulated NOX3 activity. NOXO1 alone,however, did not influence NOX3-dependent superoxide production (FIG.6B, lower panel).

At a least on a biochemical level, there is promiscuity among theorganizer and regulator subunits: NOXO1 is able to function withp67^(phox), and NOXA1 with p47^(phox) [24-26]. Therefore, weinvestigated which combinations of organizer and activator subunits arecapable to regulate NOX3, and what kind of properties those complexesmay have. Expression of NOXO1, p67^(phox), and NOX3 in HEK293 cells, ledto spontaneous superoxide generation that could not be further enhancedby PMA (FIG. 6C). However, when p47^(phox), NOXA1, and NOX3 wereexpressed, superoxide production by HEK293 cells was largelyPMA-dependent (FIG. 6C). Thus, the organizer subunit (p47^(phox) versusNOXO1) determines whether NOX3 activity is PKC-dependent or independent.

Cisplatin Enhances NOX3 Activity

Cisplatin is an ototoxic drug that exerts its toxic effect, at least inpart, through induction of ROS generation in the inner ear [2]. Wetherefore investigated the effect of this drug on NOX3 activity. HEK293cells were transfected with NOX3 or with a control vector (pcDNA3.1) andincubated for 12 hours in the presence or absence of 20 μM cisplatin.Cisplatin alone elicited superoxide production in NOX3-transfected, butnot in control-transfected cells (FIG. 7A, see traces before PMAaddition and FIG. 7C). Addition of PMA further increased superoxidegeneration, while an NADPH oxidase inhibitor, diphenylene iodonium(DPI), blocked it completely (FIG. 7A).

When HEK293 cells were co-transfected with NOX3, NOXO1 and NOXA1, theyproduced ROS in a constitutive manner (see FIG. 6B). To investigate theeffect of cisplatin under these conditions, we generated HEK293 clonesstably expressing NOX3, NOXO1, and NOXA1 subunits. These clones producedsuperoxide constitutively and spontaneously as observed in the transienttransfectants. Upon incubation with 20 μM cisplatin (12 hours), a markedincrease of superoxide production was detected by the luminol-amplifiedchemiluminescence (FIGS. 7B and C), and also by cytochrome C reduction(not shown). The superoxide generation was insensitive to PMA and couldbe abolished by DPI (FIGS. 7B and D). As control we investigated theeffect of another chemotherapeutic drugs 5-fluorouracil, which is devoidof ototoxicity; incubation of NOX3/NOXO1/NOXA1 expressing cells withthis compound (100 μM, 17 hours) did not influence superoxide production(data not shown). HEK293 cells were also co-transfected with NOX3,p47^(phox), and p67^(phox), and incubated with 20 μM cisplatin for 12hours. Cisplatin enhanced the superoxide production of NOX3-,p47^(phox), and p67^(phox)-transfected cells by a factor ofapproximately 3.3 (FIG. 7D); this superoxide production could be blockedby addition of 5 μM DPI (not shown).

Next the concentration and time dependency of the cisplatin effect onNOX3 activity was investigated using a NOX3/NOXO1/NOXA1 transfectedstable clone. After incubating the cells with various concentrations ofcisplatin for 12 hours, superoxide production was measured (FIG. 7E).Cisplatin caused an increase of NOX3-dependent ROS generation already at1 μM concentration, and 20 μM cisplatin had a maximal effect (FIG. 7E).The EC₅₀ of NOX3 activation by cisplatin was 3.6+/−1.4 μM.

In order to examine the time course of NOX3 activation by cisplatin, aNOX3/NOXO1/NOXA1 transfected stable clone was incubated with 20 μMcisplatin for various periods of time. Cisplatin enhanced NOX3 activityalready after 5 hours treatment and reached its maximal effect afteraround 17 hours (FIG. 7F); the t₅₀ was 11.5+/−1.7 hours.

FURTHER REFERENCES

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The invention claimed is:
 1. A pharmaceutical composition comprising ansiRNA inhibitor of NOX3 and a pharmaceutically acceptable carrierwherein NOX3 protein comprises or consists of the amino acid sequence ofany one of SEQ ID NOs: 1, 3, or 5, and wherein the composition issuitable for administration to the inner ear of a human subject.
 2. Thepharmaceutical composition of claim 1, further comprising an ototoxicagent.
 3. The pharmaceutical composition of claim 2, wherein saidototoxic agent is an antibiotic.
 4. The pharmaceutical composition ofclaim 3, wherein said antibiotic is an aminoglycoside antibiotic.
 5. Thepharmaceutical composition of claim 1, wherein the siRNA, represses theexpression of NOX3 protein.
 6. The pharmaceutical composition of claim2, wherein said ototoxic agent is a cytostatic.
 7. The pharmaceuticalcomposition of claim 6, wherein said cytostatic is bleomycine,bromocriptine, carboplatinum, cisplatin, methotrexate, nitrogen mustard,vinblastine, or vincristine.
 8. The pharmaceutical composition of claim4, wherein said aminoglycoside antibiotic is amikacin, gentamycin,kanamycin, neomycin, netilmycin, streptomycin or tobramycin.
 9. Thepharmaceutical composition of claim 3, wherein said antibiotic iserythromycin, vancomycin, minocycline, polymixin B, amphotericin B, orcapreomycin.
 10. The pharmaceutical composition of claim 2, wherein saidototoxic agent is a salicylate.
 11. The pharmaceutical composition ofclaim 10, wherein said salicylate is aspirin or methyl salicylate. 12.The pharmaceutical composition of claim 2, wherein said ototoxic agentis a non-steroidal anti-inflammatory agent.
 13. The pharmaceuticalcomposition of claim 12, wherein said nonsteroidal anti-inflammatoryagent is selected form the group consisting of diclofenac, etodolac,fenprofen, ibuprofen, indomethacin, naproxen, piroxicam, and sulindac.14. The pharmaceutical composition of claim 2, wherein said ototoxicagent is a quinine derivative.
 15. The pharmaceutical composition ofclaim 14, wherein said quinine derivative is selected from the groupconsisting of chloroquine phosphate, quinacrine hydrochloride, andquinine sulphate.